Mutations in mtDNA cause >200 genetic diseases. One in eight unrelated females is a carrier of an mtDNA disease. Most of mtDNA diseases are heteroplasmic - for them, the disease?associated alleles are present alongside the wild?type alleles. The severity of the mtDNA diseases depends on the frequency of the disease associated alleles in a tissue. Therefore, it is critical to study how mtDNA allele frequencies change between generations and within a human body. Such changes are governed by mutation, genetic drift, and selection. Yet the basic parameters and relative contribution of these evolutionary processes to shaping the mtDNA genetic makeup have neither been modeled nor characterized in detail. The goal of this proposal is to investigate evolutionary processes governing mtDNA allele frequency changes between generations and among tissues of an individual. To achieve this goal, we have created a unique resource - collected samples from buccal and blood cells of mother and two child (mother?2child) trios from a human population (from Central Pennsylvania). We will sequence and analyze their mtDNA with newly developed computational and statistical tools packaged in a reproducible software pipeline that will be shared with the scientific community. In Aim 1 we will develop a novel population genetics framework for mtDNA mutation and drift, and will apply it to mtDNA sequences from 200 mother?2 child trios. We will estimate germ?line, embryonic, and somatic mutation rates and bottleneck sizes. The germ?line bottleneck size estimate is needed to predict heteroplasmy levels in children, and the embryonic one to parse the distribution of heteroplasmies among tissues. The germ?line mutation rate estimate will be useful for human evolutionary studies, and the somatic mutation rate estimate will inform us about mutation accumulation in mtDNA diseases. Aim 2 will test a potential effect of age on accumulation of mtDNA mutations in the female germ line. In addition to mother?2 child trios, here we will examine female germ line directly - by sequencing mtDNA from unfertilized oocytes from 100 women of different ages. mtDNA germ?line mutation rates might increase because of oocyte aging processes which create a mutagenic environment. An age?related increase in mtDNA mutation rate, if found, will be important for formulating family planning recommendations in modern Western societies in which reproduction is frequently delayed. In Aim 3 we will develop novel likelihood?based methods for detecting selection at mtDNA. Applying these methods to the mtDNA sequencing data from mother?2child trios and unfertilized oocytes, we will contrast the strength of germ?line vs. somatic mtDNA selection, and evaluate whether mitochondrial selection operates predominantly at the level of individual mitochondria in a cell (or cells within a tissue), or amon individuals in a population. This will significantly contribute to an ongoing debate about where in an organism mtDNA selection operates. Thus, using innovative methodology, we will address pivotal questions about mtDNA evolution and disease.